Monoclonal antibodies for detection of urinary trypsin inhibitors

ABSTRACT

Certain monoclonal antibodies are able to detect urinary trypsin inhibitors (UTIs) that are characteristic of disease in humans. In particular, the UTIs include AMBK, Bikunin, Uristatin, Uristatin-1, Uristatin-2, as defined herein, also including the fragments and aggregates thereof.

Priority is claimed from PCT/US2004/024881 filed Jul. 29, 2004 whichclaims priority of U.S. provisional application No. 60/511,835 filedOct. 16, 2003.

BACKGROUND OF THE INVENTION

This invention relates generally to the detection of urinary trypsininhibitors (UTIs) in human urine. UTIs inhibit one or more of the Serineproteases. Trypsin is a member of the family of Serine proteases, i.e.enzymes, that includes trypsin, elastase, kallikrein, plasmin, thrombin,chymotrypsin, and cathepsin, among others. This group of inhibitorsprimarily forms after an increase in the number of white blood cells inthe body due to the release of elastase during infection orinflammation. UTIs are not normally found in the urine produced byhealthy individuals. The amount is elevated in those whose bodies havebacterial infections and inflammatory disorders or other maladies suchas malignant tumors, kidney disease, myocardial infarction, lungemphysema, surgical trauma, and kidney stones among others.

When infections and/or inflammation occur, the bodies' response involvesthe production of serine proteases such as elastase released byneutrophils. Non-inhibitor forms of UTI, called pro-inhibitors, such asinterleukin-α-inhibitor (I-α-I) and the pre-interleukin-α-inhibitor(P-α-I), circulate freely in plasma of healthy and diseased individuals.Serine proteases cause proteolysis of the pro-inhibitors and release thelower molecular weight UTIs into active function. The released urinarytrypsin inhibitors act on serine proteases and are later excreted in theurine. Discovered in 1909, urine trypsin inhibitors are Kunitz-typeprotease inhibitors and have been named HI-30, Mingin, Urinastatin,Serpin, and Ulinastatin over the years with the scientific communitysettling on the name Bikunin for a prevalent fragment of ˜30 Kdamolecular weight. The amino acid sequence of the Bikunin inhibitorfragment is known. It contains two Kunitz inhibitory binding domains anda large and variable chondroitin sulfate chain. See the InternationalJournal of Biochemistry and Cell Biology 32 (2000) 125-137.

Although the amount of urinary trypsin inhibitors has been measured byseveral methods, e.g. enzyme inhibition, antibody stains, latexagglutination, and radioimmunoassays, all of the UTIs in the sample aremeasured. It has not been shown that certain forms of the inhibitor canresult from chronic disease and are more easily distinguished from theinhibitors which were present in individuals with lesser degrees ofinflammation. As a result, the diagnostic use of UTIs has to date onlybeen as a non-specific marker of infection and/or inflammation. Thenon-specific nature lessens the clinical utility as determination ofUTIs does not help the care giver to know the type or site of infectionand makes it difficult to separate conditions for which the patientwould need therapy from general ailments. The present invention isdirected toward making such distinctions.

One method of measuring the UTI content of a urine sample involves theaddition to the sample of known amounts of trypsin and then measuringthe degree to which the trypsin has been inhibited. Examples of thistechnique can be found in three published U.S. Patent Applications2001/0055816 A1, 2002/0004219 A1, and 2003/0125577 A1. In thesepublished applications, a known amount of trypsin on a substrate capableof producing a detectable response is added to a sample of urine. Thesubstrate is cleaved by trypsin to yield detectable byproducts. Iftrypsin inhibitors are present, the response is diminished since some ofthe available substrate is not cleaved. Thus, by measuring the amount ofthe trypsin present and functioning relative to the amount added, theUTI content can be determined. This method detects any inhibitor oftrypsin activity and is non-specific for any given trypsin inhibitorexcreted into urine. The patent application published in 2001 disclosesthe use of a polycarboxylic chelating agent to inhibit the interferenceof calcium in the sample. The patent applications published in 2002 and2003 concern certain aromatic esters of arginine shown to be useful assubstrates for trypsin in the method just described.

Another method which determines directly the amount of UTIs in a urinesample involves the development of antibodies which attach themselves tothe urinary trypsin inhibitors and which, by the addition of immunoassayreagents containing the antibody to a sample, produce a response signal.There are various immunoassay methods which could be used to applyantibodies of the invention such as microparticle capture immunoassays(MIC), latex agglutination inhibition (LAI), solid phase chromatographic(IC), radioimmunoassays (RIA), enzyme linked immunosorbent assays(ELISA), enzyme linked assays (EIA), fluorescence linked assays (FIA),luminescence linked assays (LIA), rare earth metals label assays,chemiluminescence assays (CLA) and optical color label assays (OA) suchas colored latex particle and colloidal gold. It is also feasible to useelectrochemical signal transducers (EST) based on amperometric,impedimetric, and potentimetric detection methods.

In principle, immunoassays can be of either a heterogeneous formatrequiring a separation step or a homogeneous format without separationand either of a competitive or a non-competitive nature. Forheterogeneous assays, solid phases can be used to separate bound antigenfrom free antigen and can include plastic wells, tubes, capillaries,membranes, latex particles, and magnetic particles. Antibodies areattached to the solid phases. Antibodies can also be attached orconjugated (labeled) with reagents that directly or indirectly producedetectable responses, other antibodies or other reagents in a variety offashions. An immunoassay can also employ multiple and differentantibodies in a variety of manners such sandwich assays, double labelassays, and multiple sandwich assays depending on the detection needsfor the material being detected.

In general, the immunoassay reagents undergo changes whereby a signal isgenerated and the intensity of the signal generated is proportional tothe concentration of the analyte measured in the specimen or sample.Immunoassay reagents contain indicator dyes, metals, enzymes, polymers,antibodies, surface active agents, particles, electrochemically reactiveingredients and various other chemicals dried or filled onto carriers.Carriers often used are tubes, cups, capillaries, strips, vials, papers,microfluidic devices, cassettes, membranes or polymers with varioussample volume, uptake and transport properties.

It has been found that polyclonal antibodies produced from rabbitsinoculated with urinary trypsin inhibitors purified from the urine ofpatients with kidney disease are useful in measuring the amount of totalUTIs in urine samples from both healthy individuals and those withdisease. However, the polyclonal antibodies are not able to distinguishthe various forms of UTI from each other, in particular from thepro-inhibitors interleukin-α-inhibitor (I-α-I) andpre-interleukin-α-inhibitor (P-α-I). Since all these proteins arereadily present in the healthy patients, the cross reactivity of thepolyclonal antibody makes it useless for distinguishing diseasedpatients from healthy patients by using blood specimens. In the case ofurine specimens, the high molecular weights of P-α-I and I-α-I make itless likely that they will pass through the kidney, thus the polyclonalantibodies were more effective in spite of their reduced specificity.However due to the non-specific nature of the polyclonal antibodies theyhave not been found to be more effective than the general enzymeinhibition method previously discussed. The polyclonal antibodies werecross-reactive to all forms of UTI in urine and not specific to any oneform and would measure both inhibitory and non-inhibitory UTI.

In Journal of Immunoassay 1991; 12:347-69, Trefz et al disclose the useof a monoclonal antibody produced from mice immunized with highmolecular weight (240 kDa). Inter-α-trypsin inhibitor (an inactivepro-inhibitor) in an enzyme-linked immunoassay (ELISA). The resultsshowed that the method was effective to distinguish between healthyindividuals and those with disease, since the level of UTIs in the urineof those individuals with disease was higher than those without disease.The authors noted that Inter-α-trypsin inhibitor (ITI) having amolecular weight of about 240 kDa, contained several lower molecularweight peptides, understood to result from disintegration of the ITI.Their monoclonal antibody, IATI5, was found to recognize three majorbands, a 240 kDa, a 120 kDa, and a 50 kDa band. In a purified HI-30preparation their monoclonal antibody detected a protein of about 33kDa.

In their work reported in the Journal of Biological Chemistry, Vol. 275,No. 28, issue of July 14, pp. 21185-21191, 2000, Hirashi Kobayashi etal. compared polyclonal and monoclonal antibodies raised against apurified preparation of UTI that was shown to have molecular weight inthe range of about 40-80 kDa in SDS-PAGE and Western blotting.Derivatives were found to have molecular weights of about of 7 kDa, 30kDa, and 60 kDa.

During the cross-reactivity studies of a polyclonal antibody for UTIprepared by Bayer, we were surprised to discover that further breakdownof the inhibitory Bikunin occurs during the acute phase infections inpatients leading to the formation of other inhibitory UTIs containingboth Kunitz inhibitor domains, but lacking the chondroitin sulfatechain. This UTI, termed Uristatin, has a molecular weight of ˜17 kDa. Ina paper (Clinical Chemistry Acta (2004) 341, 73-81) reporting tests witha dipstick for detecting urinary trypsin inhibitors, Pugia et al showedthat the dipstick reported the presence of two forms for UTIs; Bikuninsand Uristatins. They identified the typical molecular weight of Bikunin(30.9 kDa) and three key forms of the Uristatin, designated Uristatin-1(5.9 kDa), Uristatin-2 (8.5 kDa) and the combination of Uristatin-1 andUristatin-2 which was termed Uristatin (17.4 kDa). All forms ofUristatin lack the chondroitin sulfate chain, and are very prevalent inpatient specimens when analyzed by electrophoresis. All Uristatin formsare inhibitory to the trypsin family of proteases; therefore theycontain at one of the two Kunitz inhibitory domains that inhibit theprotease active site upon binding. Uristatin-1 contains binding domain 2and Uristatin-2 contains binding domain 1.

It was further noted that, given the conditions of the patient used forcollection, the typical molecular weights of IUTIs could varyconsiderably. Additional variations of Bikunin and Uristatin are due tofragmentation of peptide structure, variations in the peptide sequenceand variations in carbohydrate sequences attached to the Bikunin andUristatin. Variations in molecular weight resulting from fragments occurby cleavage of the peptide sequence. A high degree of fragmentation isexpected during inflammation as the inhibitors are exposed to theproteases that can cause cleavage. Elongation and fragmentation of thecarbohydrate portions was also expected during inflammation as theglycoprotein are metabolized by a number of glycosyl transferases andglycosidases causing a great number of possible variants to thechondroitin sulfate chain attached to Bikunin and to the sugar sidechains attached to Uristatin. Additional variations also occur byaggregation of the fragments into diners or higher oligomers, especiallythrough association and metabolism of the carbohydrate portions.Therefore the functional UTI proteins represent a range of possibleproteins around a typical molecular weight.

An additional protein in this suprafamily is the precursor protein(AMBK) that initiates the biosynthesis of the inhibitory andpro-inhibitor forms. This protein is present in the plasma of patientswhen the genes signal up-regulation to initiate the biosynthesis. TheAMBK contains the Bikunin but lacks the heavy chains that inactivate theinhibitory domains and is attached to alpha-1-microglobulin. Thedetection of this form of the inhibitor is also important in determiningthe body's response to inflammation and infection.

There remains a need for a method of measuring specific urinary trypsininhibitors that is able to detect the presence of disease by determiningcertain characteristic UTIs not found in healthy individuals. Thepresent inventors have developed a method for making such determinationsby using certain monoclonal antibodies, as will be described in detailbelow.

SUMMARY OF THE INVENTION

The invention includes certain monoclonal antibodies that are able toattach themselves to inhibitory urinary trypsin inhibitors (UTI)characteristic of disease in humans and that can be detected by standardanalytical techniques, including MIC, LAI, IC, ELISA, EIA, RIA, LIA CLA,OA and EST (acronyms defined above) in both heterogeneous andhomogeneous assays. These monoclonal antibodies are characterized bytheir ability to allow direct or indirect measurement of urinary trypsininhibitors such as Bikunin, Uristatin, Uristatin-1 Uristatin-2 and AMBKin the presence of other common proteins in urine and serum such asHuman Serum Albumin (HSA), Tamm-Horsfall protein (THP),α-1-microglobulin (α-1M), α-1-antichymotrypsin (α-1ACT) andnon-inhibitory urinary trypsin inhibitors (pro-inhibitor forms) such asI-α-I and P-α-I.

Direct measurement of a UTI may be obtained by either using antibodiesthat recognize only that particular UTI. Indirect measurement of a UTImay be made by subtraction using antibodies that recognize UTIs otherthan the one of interest and antibodies that recognize all or most ofthe prevalent UTIs. In the examples below, antibodies secreted byhybridoma ATCC 421-3G5.4C5.3B6 and hybridoma ATCC 421-5G8.1A8.5C1 areshown to be suitable for determining the amount of UTIs in both urineand blood while hybridoma ATCC 420-5D11.5G8.1E4 is suitable forcorrection of cross reactivity and determining the amount of UTIs. Otherantibodies will be shown to have similar characteristics.

Such monoclonal antibodies may be made by introducing purified UTIs intomice as an immunogen. Single hybridoma clones producing only oneantibody have been created by carrying out the procedure of Kohler andMilstein. The characteristic properties of the monoclonal antibodieshave been studied, using the Surface Enhanced Laser Desorption/Ionzation(SELDI) technique, ELISA, and other immunoassay methods.

In another aspect, the invention includes methods of using the novelmonoclonal antibodies to detect and measure forms of urinary trypsininhibitors of interest, that is, those characterizing persons havingdisease. Where a monoclonal antibody is specific to an inhibitory UTI,e.g. to Bikunin, and Uristatin, or to the precursor AMBK, they can bemeasured directly. When a monoclonal antibody is able to bind to morethan one UTI, then by using more than one antibody, the content of aparticular UTI of interest may be determined by difference. The threemonoclonal antibodies referred to above have been found to bindpreferentially to Uristatin and Uristatin-1 and -2 but not to thepro-inhibitors.

Analyses may be carried out on samples of many biological fluids,including but not limited to blood, urine, water, saliva, spinal fluid,intestinal fluid, food, and blood plasma. Blood and urine are ofparticular interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar chart of the results of Example 5.

FIG. 2 is a second bar chart of the results of Example 5.

FIG. 3 is a bar chart of the results of Example 6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

Since the literature has employed various names for enzyme inhibitors,the following will provide definitions for the terms as used herein. Ithas been found that many variants have been detected in the SELDI®analysis discussed below. Consequently, it is believed that thedefinitions should encompass, not only the principal molecular weight ofthese inhibitors, but also a range of related molecular weight proteins.

Urinary Trypsin Inhibitors (UTIs) means all the inhibitors identified asinhibiting the serine proteases, including without limitation Bikunin,HI-30, AMBK, Uristatin, Uristatin-1, and Uristatin-2 including fragmentsand variants and aggregates that are capable of inhibition.

HI-30 means a fragment of higher molecular weight urinary trypsininhibitors that may or may not be identical to bikunin and aggregates.Its monomer molecular weight is reported to be 30 kDa with a range of 21to 46 kDa for fragments and variants.

AMBK means the protein precursor of pro-inhibitors which has threeconnected proteins namely the inhibitor portion (HI-30) plusalpha-1-microglobulin plus a 25 kDA unidentified protein and has amolecular weight of about 66 kDa with chondroitin sulfate and 42 kDawithout chondrotin sulfate, in range of 42 to 70 kDa. It is consideredto be within the definition of UTI for purposes of the invention,without regard to its function in the biosynthesis of inhibitory serineproteases.

Tamm-Horsfall glycoprotein means the glycoprotein also known asuromucoid commonly found in urine and having a typical molecular weightof about 85 kDa with a range of 80 to 91 kDa and including the variousfragments and variants of.

Bikunin means a protein consisting of one or two proteinase inhibitordomains of the Kunitz type that can be connected by a short peptidechain and extended by N- and C-terminated polypeptide chains. Theprotein is linked to a sulphated chondroitin chain and to anoligosaccharide and has a molecular weight of about 33 kDa (range of21-46 kDa). The structure is shown by Pugia et al in ClinicalBiochemistry 32 (2002) 105-110 and the 2000 paper by Fries and Blom inthe International Journal of Biochemistry & Cell Biology discussedabove. The calculated molecular weight of Bikunin is 30.9 kDa in thepresence of both the chondroitin sulfate chain (13.5 kDa) and the sugarside chains (1.9 kDa); these values are based on previously publishedsequences for HI-30.

Uristatin means a protein fragment of Bikunin resulting from cleavingthe chondroitin sulfate chain from Bikunin and having a monomericmolecular weight of about 17 kDa with a range of 11 to 22 kDa forfragments and variants. The calculated molecular weight of Uristatin is17.4 kDa lacking the chondroitin sulfate chain (13.5 kDa); these valuesare based on previously published sequences for HI-30.

Uristatin-1 and -2 mean protein fragments of Uristatin which containeither of the Kunitz type inhibitor domains 1 or 2 and have a monomermolecular weight of about 6 kDa with a range of 2 to 9 kDa for fragmentsand variants and about 8.5 kDa with a range of 2 to 12 kDa for fragmentsand variants respectively. The calculated molecular weight ofUristatin-1 and -2 are 5.9 kDa and 8.5 kDa respectively; the values arebased on previously published sequences for HI-30.

Pro-inhibitors mean the non-inhibiting form of the protein precursor ofUrinary trypsin inhibitors, including without limitation such asproteins such as I-α-I and p-α-I. These forms are considered inactiveUTIs as their measured inhibitory activity is several fold less thanBikunin and Uristatin forms. They are present in healthy and infectedpatients.

I-α-I means Inter-α-inhibitor, understood to refer to Bikunin attachedto two heavy chains, typically H₁ and H₂, by means of the chondroitinsulfate chain with a typical molecular weight of 180-240 kDa. P-α-Imeans Pre-α-inhibitor, understood to refer to Bikunin attached to oneheavy chain, typically H₃ with a typical molecular weight of 125 kDa.The inter-alpha-inhibitor and pre-alpha-inhibitor group are distinctassemblies of Bikunin with heavy chains from a distinct set of four ormore heavy (H) chains designated H1, H2, H3, H4, and etc. These H chainsare encoded by a set of evolutionarily related H genes, and bikunin isencoded by an alpha-1-microglobulin/Bikunin precursor gene (AMBK).

Monoclonal Antibodies

Monoclonal antibodies are distinguished from polyclonal antibodies inbeing identical and capable of attaching themselves to a single epitopeof an antigen. Polyclonal antibodies, in contrast, are not identical andtherefore are not able to provide the precise analysis for whichmonoclonal antibodies are valuable. Monoclonal antibodies may beproduced by the method of Kohler and Milstein, Nature 256:495 (1975). Animmunogen (antigen) of interest is injected into mice and the B-celllymphocytes produced in response to the immunogen are harvested after aperiod of time. The B-cells are combined with myeloma cells obtainedfrom mice and introduced into a medium which permits the B-cells to fusewith the myeloma cells, producing hybridomas. These fused cells(hybridomas) are then placed in separate wells in microtiter plates andgrown to produce monoclonal antibodies. The monoclonal antibodies aretested to determine which of them are suitable for detecting the antigenof interest. After being selected, the monoclonal antibodies can begrown in cell cultures or by injecting the hybridomas into mice.

Making and Separating Preferred Monoclonal Antibodies

Mice are injected with purified UTIs as the analytical target. Antibodyproducing cells are taken from the animals. Antibody-producing cells arefused with cells that grow continuously in culture to form hybridomas. Asingle hybridoma produces only one antibody. A single hybridoma dividesto produce a large population of ‘clones’ all making the same“Monoclonal” antibody. Living hybridomas are frozen indefinitely inliquid nitrogen.

The particular procedures used by the present inventors to preparemonoclonal antibodies are described in detail in the examples. It willbe understood by those skilled in the art that those procedures can bemodified or augmented and that they are not to be considered to limitthe scope of the invention. For example, other techniques for bothimmunization and fusion protocols which yield hybridomas are familiar toworkers in the field of monoclonal antibodies.

The present invention involves monoclonal antibodies that were selectedon the basis of their ability to bind to one or more UTIs and alsopreferentially to bind to certain epitopes on the intact molecule whichthen appear on particular fragments once the break-down of the UTIsoccurs.

Detecting Trypsin Inhibitors in Biological Fluids, e.g. Urine

Once the selective monoclonal antibodies have been located, they may begrown by conventional procedures and used in ELISA testing, or any otherimmunoassay technique described earlier to detect the presence of UTIsthat are characteristic of infection and/or inflammation.

ELISA (enzyme-linked immunosorbent assay) provides a very sensitivemethod for detecting antigens. In an antigen down ELISA, a microtiterplate receives a sample suspected of containing a certain antigen. Afterallowing for adsorption of the antigen onto the plate, and washing offall non-bound materials, the monoclonal antibody is added, incubated sothat it can bind to the antigen and the excess washed off. The addedmonoclonal antibody may have already been labeled with a reportermolecule to permit the generation of a signal to be read by any numberof techniques. Alternatively a ligand capable of attaching to theantibody (e.g. an anti-mouse antibody conjugated to a reporter molecule)is added. After excess of the enzyme-coupled ligand has been washed off,the chromogen or other substrate is added if necessary and the colordeveloped used as an indicator of the amount of antigen present.

There are various immunoassay methods that could be used to applyantibodies of the invention such as microparticle capture immunoassays(MIC), latex agglutination inhibition (LAI), solid phase chromatographic(IC), radioimmunoassays (RIA), enzyme linked immunosorbent assays(ELISA), enzyme linked assays (EIA), fluorescence linked assays (FIA),luminescence linked assays (LIA), rare earth metals label assays,chemiluminescence assays (CLA) and optical color label assays (OA) suchas colored latex particle and colloidal gold. It is also feasible to useelectrochemical signal transducers (EST) based on amperometric,impedimetric, and potentimetric detection methods.

EXAMPLE 1 Preparation of Monoclonal Antibodies

BALB/c mice were immunized with 100 μg/mouse of purified UTIs obtainedfrom renal patients by SciPac Ltd. Sittingbourne, Kent, UK, product codeP250-1 to produce a response. After one month, ocular bleeds were takenfrom each mouse and titered by ELISA against the immunogen to assess theimmune response. The mice showing the best response were boosted byinjection of 100 μg/mouse with the immunogen. After four days, mice weresacrificed and their spleens used for fusion according to the method ofKohler and Milstein, Nature 256:495 (1975). The spleenocytes were fusedwith SP2-0 Ag14 myeloma cells using PEG (polyethylene glycol) solutionwith a ratio of spleenocytes to Myeloma cells of 5:1 and plated into 96well plates using 50% PEG/HAT growth media. After 7-10 days ofincubation at 37 degrees Celsius, fusion cultures were monitored forgrowth by feeding every 3-4 days utilizing the HAT (hypoxanthine,aminopterin, thymidine) selection method followed by subculturing withHAT growth media.

After 2-3 weeks, the wells having hybridoma colony growth were tested byELISA to determine which growths produced an antibody immune response tothe uristatin peptide. The 96 well plate cultures were tested with theuristatin peptide at 1 ug/mL coated plates. After coating platesovernight at 2-8° C., all plates were washed and blocked. Cell culturesupernatants were then applied 100 μl/well for one hour at roomtemperature. After washing plates, Goat anti-mouse IgG Horse RadishPeroxidase at 1:2000 dilution was applied at 100 μL/well for one hour.Plates were washed once again followed by OPD (o-phenylene diaminedihydrochloride) substrate and read at 490 nm on a Spectra Max® platereader.

The colonies giving a positive response were transferred to 24 wellplates for further expansion and retesting to verify the positiveresults. The colonies testing positive were further expanded in six wellplates in Iscove's Modified Dulbecco's Medium (IMDM) with 10% FetalBovine Serum (FBS). After expansion, the colonies were frozen at −70° C.and then transferred to liquid nitrogen for long-term storage.

Based on ELISA results using the purified UTIs, various clones werefurther expanded in IMDM, 10% FBS and frozen down.

EXAMPLE 2 Screening Procedure of Polyclonal Antibodies

Rabbit polyclonal antibodies raised against the purified UTIs were usedin the screening process as this antibody was expected to benon-specific for any given form of UTIs. Serum and urine specimens fromthree patients with infection and two healthy controls werecharacterized by western blot tests using these polyclonal antibodies.The western blot tests used the commercial pre-cast gel system(Invitrogen, San Diego Calif.). Urine and plasma specimens were loadedwith 1 μg and 5 μL per lane. The western blots were stained with aWesternBreeze® chromogenic immunodetection kit (from Invitrogen)following the manufacturer's instructions. Rabbit anti-UTI antiserum wasused at a dilution of 1:250 000 as the primary antibody in the westernblot analysis (See Table 1). These western blot results demonstratedthat the rabbit polyclonal antiserum body detected Uristatin, Bikunin,AUBK, TBP, P-α-I, and I-α-I. The THP cross reactivity was unexpected andlikely due to a common peptide sequence between the urinary trypsininhibitors and THP.

TABLE 1 Results of western blot analysis using polyclonal serum asinterogating antibody of specimens Major proteins Minor proteins Majorproteins Minor proteins observed in observed in observed in observed inSpecimen urine urine plasma plasma Patient 1 Uristatin Bikunin, AMBK,P-α-I, I-α-I, AMBK, Bikunin THP Patient 2 Bikunin Uristatin, AMBK,P-α-I, AMBK, Bikunin THP Patient 3 Bikunin Uristatin, AMBK, P-α-I,I-α-I, AMBK, THP Bikunin Healthily control 1 None THP P-α-I, I-α-I, AMBKHealthily control 2 Bikunin THP, P-α-I, I-α-I, P-α-I, I-α-I, AMBK,Bikunin AMBK and THP

These western blot tests also demonstrated that Uristatin was betterable to differentiate diseased patients from healthy controls thanBikunin, P-α-I, I-α-I, AMBK and THP. All patients and controls had THPin the urine. Similarly all had P-α-I, I-α-I, and AMBK in the plasma.The results showed that Bikunin was found in the urine of the threepatients with infections but also in the urine of one control subject.We found Uristatin in all three patients but not any of the controlsubjects. Also we found marked increase of Uristatin in one patient. Theresults showed that AMBK was found in the urine of the three patientswith infections but also in the urine of one control subject. Thissupports our belief that specific forms of UTIs can be more related todisease in certain patients than others. It should be noted here thatBikunin and especially Uristatin are in much lower concentration inblood than the pro-inhibitors I-α-I and P-α-I because they pass readilyinto the kidneys. The pro-inhibitors do not pass through the kidneysinto urine in significant amounts, making it easier to detect Uristatinand Bikunin in urine than in blood. THP is excreted in the proximaltubules of the kidney, therefore not required to pass through thekidney, and is the most common protein in the urine as evidenced bypresence in patients and controls tested. The high cross reactivity ofthe polyclonal method does not allow detection of the lowerconcentrations of Bikunin or Uristatin in blood or urine.

The presence of urinary trypsin inhibitors has been shown to bemeaningful in a number of diseases. These disease include infection(pneumonia, urinary tract infection, upper respiratory tract infection,neonatal sepsis, meningitis, appendicitis, bacterial infections, boneand joint infections), acute inflammation (trauma, kidney stones,surgical truama, cardiopulmonary bypass, myocardial infarction, andburns), chronic inflammation (rheumatoid arthritis, lung emphysema,inflammatory bowel disease, pancreatitis, Crohn's disease, and as a riskfactor for cardiovascular disease), neoplasia (leukemia, multiplemyleoma, non-hodgkins lymphoma, ovarian cancer, breast cancer,pancreatic cancer, stomach cancer, colon cancer), and kidney disease.

Clinical value is assessed by the ability of a method to detect theurinary trypsin inhibitor forms more prevalent in disease. Also methodsable to work not only in urine but also in blood would be of additionalclinical value. Blood is the preferred fluid for clinical laboratoryanalysis allowing consistency in results, while urine is the preferredfluid for allowing non-invasive sampling at the point of care.

Therefore, an immunoassay which detects only specific forms of UTI's,such as Uristatin, Bikunin, and AMBK but not P-α-I, I-α-I or THP wouldhave clinical utility. The results of our tests also showed that nodetectable P-α-I or I-α-I was in urine but was present in all plasmaspecimens from controls and patients. Any immunoassay of blood foractive UTIs such as Uristatin, Bikunin and AMBK should not detect P-α-Ior I-α-I. The predominant bands in blood corresponded approximately toan AMBK of 63 kDa, P-α-I of 115 to 125 kDa, and I-α-I of 220 kDa in bothpatients and controls.

All the monoclonal antibody clones were screened based on their abilityto bind UTIs in the ELISA assay using the procedure described in Example1 and the patterns observed in western blot tests for antibodies usingpatient and healthy specimens as described in Table 1. The antibodieswere grouped by similarity of patterns into three groups and the bestrepresentative antibodies of each group selected based on ELISA testingagainst the UTIs standard. The selection of clones for further studywere as follows, from group A: 421-305, from group B: 421-508, and fromgroup C: 420-5D11. For the selected mother colonies, cell culturesupernatants were cloned out twice using the limiting dilution methodassure that the antibodies were monoclonal. The hybridomas secretingthese monoclonal antibodies in Iscove's Modified Dulbecco's Medium(IMDM) 30% Fetal Bovine Serum (FBS), and 10% Dimethyl Sulfoxide (DMSO)were deposited at the American Type Culture Collection, Mannassas, VA20110-2209, USA on Jul. 28, 2006 and designated as ATCC421-5D11.5G8.1E4, ATCC 421-3G5.4C5.3B6, and ATCC 421-5G8.1A8.5C1. Thepatent Deposit Designations are PTA-7744, PTA-7746, PTA-7745respectively. The receipt comfirms the deposited materials will beavailable once a U.S. Patent has issued.

EXAMPLE 3 Characterization of Monoclonal Antibodies Against PurifiedUristatin and Bikunin Standards

The urinary trypsin inhibitors were produced by SciPac Ltd for our study(Sittingbourne, Kent, UK, product code P205-1); these are purified fromthe urine of patients with chronic renal failure. The standards werecharacterized by SDS-PAGE. We used a commercial pre-cast gel system(Invitrogen, San Diego Calif.) 4-12% NuPAGE® Bis-Tris with a MES(2-morpholinoethanesulfonic acid) running buffers (reducing andnon-reducing), following exactly the manufacturer's procedure. Specimenswere loaded at 2 μg per lane. Estimations of the proteins' molecularweights were based on a full set of standards: MagicMark12™ (Invitrogen)and SeeBlue®Plus2 (Invitrogen). Protein bands were stained withColloidal Blue® (Invitrogen).

The results of these tests showed that UTI lot 20-120 contained 15-20%17 kDa proteins, 50-55% 35 kDa proteins, and 25-30% 60-80 kDa proteins.UTI lot 124-111 had about 85% of the material as the 17.4 kDa band(Uristatin) but also contained amounts of Uristatin-1 (5.9 kDa) and10-15% of Bikunin (30.9 kDa) but no AMBK was seen. UTI lot 80-117contained substantially only 17 kDa Uristatin material.

These initial results were further refined by the SELDI® techniquediscussed below. It was found that UTI lot 20-120 contained 10% of 2-12kDa proteins (Uristatin 1 & 2), 15% of 17 kDa proteins (Uristatins), 45%of 35 kDa proteins (Bikunin), and 30% of 60-80 kDa (AMBK or THP)proteins. UTI lot 124-111 had about 80% of the material as the 17.4 kDaband (Uristatin) but also contained amounts (10%) of Uristatin-1 and 2(5.9 kDa) and 10% of Bikunin (30.9 kDa) but no AMBK was seen to bepresent. UTI lot 80-117 containing substantially only the 17 kDaUristatin material and less than 2% of other molecular weight ranges.

The urine samples for the controls were assayed by the ELISA assaydescribed in Example 1 using the three monoclonal antibodies selected inExample 2 and a Goat Anti-Mouse secondary antibody conjugate. Theresults for group A clone 421-3G5, group B clone 421-5G8 and group Cclone 420-5D11 are shown in Table 2 and compared to the polyclonalantibody as a control.

The measurements made of color developed by the OPD substrate indicatedthe amount of the monoclonal antibody bound to UTIs in the standardsjust described, whose contents were determined by molecular weightsusing the western blot method and SELDI®. The values were normalizedusing the polyclonal antibody values and are presented in the tablebelow as percent relative to the polyclonal antibody results.

TABLE 2 ELISA Results with Specimens from Patient and Healthy Individualand Protein Standards for UTIs. ANTIBODY ANTIBODY ANTIBODY GROUP B GROUPC GROUP A 2–12/17/35/ CLONE CLONE CLONE POLYCLONAL Specimen 60–80 kDa %421-5G8 420-5D11 421-3G5 ANTIBODY UTI Standard 10/15/45/30  92% 100% 71%100%  lot 20-120 UTI Standard 10/80/10/0 100%  13% 100%  94% lot 124-111UTI Standard 0/100/0/0  5%  0% 48% 74% lot 80-117

The monoclonal antibody 421-5G8 bound strongly to UTI lots #124-111 and#20-120 to a similar degree, but only bound weakly to Lot #80-117. Thiswould be consistent with binding to the 2-12 kDa material (Uristatin 1or 2) in both lots but not-in lot 80-117 containing only the 17 kDamaterial (Uristatin). Thus, this antibody appears specific for Uristatin1 or 2 over Bikunin and Uristatin. While not wanting to be limited to amechanism, it is believed that binding by this monoclonal antibody doesnot occur through the sulfated chondroitin chain. The binding is thoughtto be direct to an inhibitory amino acid sequence.

The monoclonal antibody 420-5D11 bound strongly to UTI lot #20-120, veryweakly to lot #124-111, and did not bind to lot 80-117. This isconsistent with binding to the 80 kDa material (THP) in lot #20-120,since only lot #20-120 contains a large amount of this material. If theantibody were specific for Uristatin or Bikunin also, one would expectit to have strongly responded to lot #124-111, which contained about 65%of Uristatin. This antibody appears to bind to THP but also to Bikuninand Uristatin. While not wanting to be limited to a mechanism, it isbelieved that binding by this monoclonal antibody does not occur througha common amino acid region between THP and Bikunin or Uristatin.

The monoclonal antibody 421-3G5 bound strongly to all UTI lots andsimilar to the non-specific polyclonal antibody with the notableexception of lacking reactivity to P-α-I and I-α-I. Thus 421-3G5 wouldbe a measure of total UTI.

EXAMPLE 4 Testing of Monoclonal Antibodies with Clinical Specimens

Urine samples were obtained from patients having bacterial infectionsand from control patients having no such infections. The patientswithout infections are the “group 1” patients. To be included in thestudy, we only required a negative urine and blood culture (10⁵organisms/mL) and a normal blood white blood count (CBC). The secondgroup of patients (“group 2”) included those with infections eitherupper respiratory tract or urinary tract, a conclusion based on apositive complete blood count (CBC) in all. Clean-catch midstream urinecollections were obtained from all patients and controls. Specimens werestored at 4° C. until tested; but if not tested within 24 hours, storagewas at −70° C. until tested. For all subjects, the evaluation of urinesediment, gram stain and urine microbiological culture were alwaysperformed on the day of collection. We collected EDTA-anticoagulatedblood from group 2 and performed a CBC, a high sensitivity CRP (DadeBehring, Immunoassay for C-reactive protein) test, and a blood cultureand performed all these tests on the day of blood collection.

The urine samples were also assayed by an ELISA assay using the samepolyclonal antibodies as in Example 2. The antibodies were immobilizedin polystyrene membrane wells of the high binding microtiter plates (PN3690 Corning Life Sciences, Acton, Mass.), then wells were coated withSuper Block® (Pierce Chem Co., Rockford Ill.) to ensure none of thefollowing additions attach directly to the plate, then contacted with aurine sample from each patient to bind the antibodies to the UTIs in thesample. The UTI-antibody complex was reacted with a second antibody(goat anti-rabbit antibody) conjugated to alkaline phosphatase, then theunbound conjugated antibody was washed away with TBS/2% TWEEN-20 (ELISAWash buffer, Upstate Cell Signaling Solutions, Lake Placid N.Y.) and theimmobilized antibody assayed by determining the alkaline phosphatase onthe plate by hydrolysis of PNPP (p-Nitrophenyl Phosphate, Disodium Salt)forming an absorbance at 405 nm (yellow) color upon addition ofDiethanolamine Buffer (Pierce Chem Co.). The absorbance was measured aspectrophotometric micro titer-plate reader (SpectroMax.® MolecularDevices Corporation, Sunnyvale, Calif.).

It was found that three control urine samples contained 51.4, 27.9 and24.8 μg/mL of UTI proteins (Bikunin, Uristatin, and AMBK), while twosamples from patients having bacterial infections contained 148 and 49.5μg/mL. This result confirms the previous western blot test findings thatpolyclonal antibody gave poor separation of patients with infectionsfrom healthy controls as would be expected by the cross-reactivitybetween P-α-I, I-α-I and THP proteins and the desired measured proteinsBikunin, Uristatin, and AMBK.

Measurements of UTI's in the urine and plasma of patients can be madespecific by the direct method and the indirect subtraction method usingmonoclonal antibodies, such as those shown in Table 2. As an example ofdirect method, Uristatin 1 or 2 in either a urine or blood specimen canbe measured directly with clone 421-5G8 over the other UTI, THP andpro-inhibitors present. An additional example of direct method, all ofthe UTIs (IUTI) in either a urine or blood specimen can be measureddirectly with clone 421-3G5, but not the pro-inhibitors. An example ofindirect method would be subtraction of one or more specific resultsfrom a total measurement of UTI. For example, to measure the Bikunin inpatient 1's urine, Uristatin 1 or 2 would be measured directly withclone 421-5G8 and would be subtracted from a total IUTI 421-3G5 toarrive at the amount Bikunin and AMBK. Additionally any cross-reactivityto THP can measured directly with clone 420-5D11 and could be subtractedfrom an UTI antibody with cross reactivity to THP, such as thepolyclonal antibodies, to arrive at the amount of UTI. A totalmeasurement could be made by the polyclonal antibody as discussed above,monoclonal antibody clone 421-3G5 or the enzyme inhibition method. Inthe case of the polyclonal antibody, it was shown that this total wasaffected by the pro-inhibitor amounts, therefore the monoclonal antibodyclone 421-3G5 or enzyme inhibition method would be preferred or acorrection by subtraction of a pro-inhibitor result could be used.

EXAMPLE 5

In Example 3, three monoclonal antibodies were tested for their responseto three lots of purified UTIs. This example reports the same monoclonalantibodies, along with an additional sixteen monoclonal antibodies. Theresults are shown in FIGS. 1&2, a series of bar graphs.

The same three lots of purified UTIs were used. UTI lot 20-120 contained10% of 2-12 kDa proteins (Uristatin 1 & 2), 15% of 17 kDa proteins(Uristatins), 45% of 35 kDa proteins (bikunin), and 30% of 60-80 kDa(AMBK or THP) proteins. UTI lot 124-111 had about 80% of the material asthe 17.4 kDa band (Uristatin) but also contained amounts (10%) ofUristatin-1 and 2 (5.9 kDa) and 10% of Bikunin (30.9 kDa) but no AMBKwas seen to be present. UTI lot 80-117 containing substantially only the17 kDa Uristatin material and <2% of other molecular weight ranges.

It can be seen that the lot containing substantially only 17 kDamaterial was generally bound less strongly than those lots thatcontained low and high molecular weight species. However, additionalmonoclonal antibodies were found to bind all three lots in a mannersimilar to that of the polyclonal antibody control (for example421-1D11, 421-5B9, and 421-2D5 and would be expected to be similar to421-3G5. Several antibodies were found to bind the three lots similarlyto 421-5G8, for example 421-1C10, 421-1C12, 421-2A11, and 421-2G4. Thenotable exception was again confirmed with monoclonal antibody 420-5D11, which showed an affinity for lot 20-120, but not for lots 124-111and 80-117. It can be concluded that monoclonal antibody 420-5 D11 didnot bind strongly to the lower molecular weight uristatin (17 kDa) andbikunin (35 kDa) but preferred the higher molecular weight material(60-80 kDa THP and AMBK are in this range). 420-5D11 was the onlymonoclonal antibody from the fusion designated 420 (the others werederived from fusion 421). Its distinctive behavior suggested furthertesting of monoclonal antibodies, which is reported in Example 6.

EXAMPLE 6

Nine monoclonal antibodies from fusion 420 were tested in addition to420-5D11. Five lots of UTI's were used. Lots 20-120, 124-111, and 80-117were included again and lots 93-90 and 157-90 were added. The resultsshown in FIG. 3 show that monoclonal antibody 420-5D11 was similar to420-1B7, 420-104, and 420-4E11, The other monoclonal antibodies showedsubstantially no binding of the UTIs. The control used in theseexperiments was a monoclonal antibody from fusion 421 (421-5B9) that hadbeen found to have a binding profile similar to the polyclonalantibodies but without pro-inhibitor binding, that is, the monoclonalantibody of 421-5B9 binds to each of the UTI lots strongly.

EXAMPLE 7

Two samples of purified total UTIs used in the previous ELISA tests andtwo samples from patients were reacted with the three representativemonoclonal antibodies and one polyclonal antibody and analyzed todetermine the proteins binding to the antibodies, using Surface EnhancedLaser Desorption/Ionization (SELDI®) technology (PBS II SELDI® massspectrometer from Ciphergen, Fremont, Calif. Binding was measured on twotypes of surfaces using a standard incubation procedure (High BindingPS20 Chip and a low binding RS100 Chip). The signal to noise (S/N) wasdetermined for each mass bound to the antibody as a measure ofsignificance.

Also analyzed by mass spectroscopy were two urine samples from patient 4and 20 patient 5 reacted with antibodies. Both patients were affected byinflammation with elevated white blood cells (by complete blood cellcount), C-reactive protein (by high sensitivity immunoassay),ESR(erythrocyte sedimentation rate), urinalysis (by ten panel MULTISTIXPRO® dip strip) and total urinary trypsin inhibitors (by inhibitorassays). Patient 5 was positive for a blood bacterial infection(toximia) and patient 4 was positive for a urinary bacterial infection(Urinary tract infection) both by microbiological cultures.

The procedure followed is outlined as follows:

1. Three μL of 50 mM NaHCO₃ (pH 8.0) was added to each spot on theprotein chip, which was covered by a plate to form sample wells.

2. One μL of 1 mg/mL of the antibody was added to each spot andincubated at room temperature for 2 hours with shaking in a humiditychamber.

3. The solution was removed from each spot and the spot was washed twicewith 5 μL of washing buffer, phosphate buffered saline (PBS+0.5% Tritondetergent).

4. The unbound sites were blocked with 5 μL or 2 mg/mL BSA (bovine serumalbunim) or 1 M ethanolamine.

5. After incubation at room temperature, the BSA or ethanolamine werediscarded.

6. The spots were twice washed with 5 μL of washing buffer (PBS+0.5%Triton).

7. Five μL of PBS was added to each spot and the chips were placed in abioprocessor. An additional 10 μL of PBS was added in each well.

8. Ten μL of the sample to be tested (or PBS as a control) was added toeach well, then the wells were sealed and shaken at 4° C. overnight.

9. The wells were washed twice with washing buffer and PBS and thenshaken at room temperature for 2 minutes.

10. The wells were rinsed twice with 300 μL of deionized water saturatedwith sinapinic acid.

11. The chips containing the antibodies and samples were analyzed bysurface enhanced laser desorption/ionization (SELDI®).

The SELDI® results for the four antibodies are reported in the Table 3and 4. The results are the masses of proteins bound to the antibodiesafter exposure to the UTIs in the patients and purified samples. Thehigh sensitivity of this method demonstrates antibody binding to a greatmany more forms than were detected by the ELISA method. The abundance offorms is expected from the fragmentation, elongation and aggregationthat is responsible for the molecular weight ranges for the functionalclasses of UTI. This method identified exactly the forms bound and doesnot rely on any interpretation by the composition of the standardTherefore even though protein bound might be a small percentage of thecomposition, it will be detected if there is binding to the antibody.Antibody binding epitopes are typically small, on the order of 2 to 5kDa, when antibodies bind to the peptide or glycoprotein sequence infavor of the tertiary structure of the protein sequences. Therefore acommon sequence would be expected to be repeated in all variations andthe ability of SELDI® to detect all binding events allows a great manyvariations to be demonstrated. Results from all samples are combined andorganized by functional protein groups according to the observedmolecular weight falling within ranges for Uristatin 1 or 2, Uristatin,Bikunin, AMBK, THP and the pro-inhibitors. Those binding events of highaffinity, frequency or importance are in bold face as determined bysignal-to-noise ratios of binding events. The primary binding events arein bold face and underlined. Weak binding events are in plain text todocument variations in the proteins. These represent very weakcross-reactivities of the type that would not impact an immuno-assay ifproperly formated.

TABLE 3 SELDI ® Results Comparison of Three Novel Monoclonal Antibodiesfor UTIs versus a Standard Polyclonal Antibody for a total measurementof all UTI and pro-inhibitors using purified UTI standards. UTIFunctional Group polyclonal 1Mab 421-3G5 Mab 421-5G8 Mab 420-5D11 RbAnti-Uristatin IUTI Uristatin THP IUTI and clone clone clone Pro-UTIUristatin 1 or 2 - (average 3.48, 3.86 3.48, 3.86 3.48, 3.86 3.49, 3.86molecular weights of 5.9 & 8.5 kDa with kDa range 2-12) Uristatin 15.9,17.8, 15.9, 18.3 15.9, 16.3 18.2 15.9, 17.8 (17 kDa, range 11-22) 16.3,18.0 Bikunin No significant No significant 41.5, 48.1 42.5, 42.6 (33kDa, range 21-46) peaks peaks AMBK 62.9 , 62.5, No significant 67.1 66.6, 66.7 (66 kDa, range 42-70) peaks THP 81.9 81.7 81.7 82.6 (85 kDa,80-91 kDa) p-alpha-I No significant 122.6 No significant 108.6, 120.0,(125 kDa) peaks peaks 142.7 I-alpha-I No significant No significant Nosignificant No significant (220 kDa) peaks peaks peaks peaks

TABLE 4 SELDI ® Results Comparison of Three Novel Monoclonal Antibodiesfor UTIs versus a Standard Polyclonal Antibody for a total measurementof all UTI and pro- inhibitors using purified patient samples. IUTIFunctional Group Mab 421-3G5 Mab 421-5G8 Mab 420-5D11 polyclonal IUTIUristatin THP Rb Anti-Uristatin clone clone clone IUTI and Pro-UTIUristatin or 2 - 2.82, 3.41, 2.82, 3.41, 3.48, 2.82, 3.0, 3.5, 2.8, 5.0, 5.4, 7.2, (average molecular 3.48, 4.0, 5.4, 7.2, 4.7, 3.7, 5.4, 10.7,3.5, 4.0, 5.9 weights of 5.9 & 8.5 I kDA 5.38, 5.57 9.18 5.6, 7.2, 3.7,6.0, 6.3, 8.1, 10.7 with kDa range 2-12) 6.30, 7.24, 9.85 10.8 3.5, 3.8,4.0, 5.6, 6.0, 6.3, 8.0, 9.2, 9.8, 10.9 Uristatin 11.8, 18.0, 12.0,14.0, 13.5, 11.9 , 13.5, 21.1, 13.4, 14.0, (17 kDa, range 11-22) 16.3,13.4, 21.1 16.3 14.0 Bikunin 21.1, 33.6, 23.0, 22.7 23.3, 41.6 21.2,22.6, 23.3, (33 kDa, range 21-46) 35.2, 33.5, 33.4 , 33.1 35.2, 45.942.3 AMBK 67.0 No significant No significant 58.6 (66 kDa, range 42-70)peaks peaks THP 81.9, 91.1 No significant 80.2, 82.6, 79.2 (85 kDa,80-91 kDa) peaks 80.6 81.9 p-alpha-I (125 kDa) No significant 128.5,132.1 No significant 101.1, 103.7, peaks peaks 106.3, 120.0, 1234,133.0, 142.7 I-alpha-I (220 kDa) No significant No significant Nosignificant No significant peaks peaks peaks peaks

Mass differences were rounded to nearest 0.1 kDa. Peaks with highestSignal to Noise ratios (S/N) relative to other peaks are reported. LowS/N observations were eliminated from table. The table represents mostbut not all possible forms. Spectra also showed peaks at 66.9 to 66.6and 33.3 and 133.0 to 133.9 that were due to albumin used in theblocking procedure for the SELDI analysis. When triethanolamine was usedto block chips those peaks were not observed.

The results demonstrated that the three types of monoclonal antibodiesdetect different patterns of UTIs and these patterns include a range ofvariants. The monoclonal antibodies are specific for UTIs whereas thepolyclonal antibodies detect both UTIs and Pro-inhibitors. We can notsee all the urinary trypsin inhibitor fragments that the clones detectin the patterns represented in the SDS gels. The gel method is notsensitive enough to report all of the UTIs the antibody can bind. Moreof the fragments actually bound are shown in the SELDI® data. Allfragments bound are not listed as additional variations would be foundas different patients are tested. The extent or strength of binding to agiven UTI was estimated relative to other UTIs. The strongest binding bysmallest fragments are the best representation of the epitopes bound byeach antibody. The low molecular weight sequences in the Uristatin 1 or2 functional groups are the smallest linear sequence with strongbinding.

In Tables 3 and 4 using purified standards and patients, the primarybinding for Mab 421-3G5 was strong for Uristatin-1 or -2, Uristatin,Bikunin, and AMBK and much less strong for THP with no significantpro-inhibitor binding. The primary binding for Mab 421-5G8 was withUristatin-1 or -2, less for Uristatin and much less strong for THP orBikunin or pro-inhibitors. The primary binding for Mab 420-5D11 wasstrong for THP, Uristatin, and Uristatin-1 or -2, less strong forBikunin or AMBK. The polyclonal binding was with all forms including thepro-inhibitors. These results were as expected based on SDS gels. TheSELDI® results however also demonstrated that all three antibodies boundto Uristatin-1 or -2 in the standards very strongly at 3.9, and withhigh affinity at 2.8 and 3.5. The SELDI® results also show several lowaffinity binding events, not strong enough to hinder specificity, butmeasurable.

The results in Table 4 using individual patient samples, demonstrate thesame 25 primary binding patterns for Mab 421-3G5, Mab 421-5G8, Mab420-5D11 and the polyclonal shown with the standards. Again theseresults were as expected based on SDS gels. The SELDI® results howeveralso demonstrated that all three antibodies strongly bound to severalnew forms of Uristatin-1 or -2, Uristatin, Bikunin and AMBK within theexpected ranges for each. In particular primary binding to 2.8, 5.4,7.2, 11.8, 21.1, and 67.0 kDa were observed in patient samples but notin the purified standards. This demonstrated the variations expected inpathological conditions for reasons previous stated. The lack of thesepeaks in the purified standards is the result of the pooling of manypatient and purification of specimens to the target molecular weighttargets of 15, 30 and 60 kDa. The SELDI® also showed at least somecross-reactivity to THP in the polyclonal and Mab 420-5D11 case.

EXAMPLE 8

Four proteins, Human Serum Albumin (HSA), Tamm-Horsfall protein (THP),α-1-microglobulin (α-i M), and α-i-antichymotrypsin (α-i ACT) weretested for crossreactivity in the Uristatin sandwich ELISA test. Theseproteins were chosen either because of their high concentration in urineor because they were suggested as possible cross reactants during thewestern blot or SELDI® work that used these antibodies with urinesamples. Cross reactivity studies were done in a competitive format toallow relative binding to be compared. For example the binding ofantibodies to uristatin was significantly stronger than to THP, theassay for uristatin was not interfered with by THP. The only proteinsand antibodies found to be cross-reactive in the competitive sandwichELISA test was THP with Mab 420-5D11 and the polyclonal antibody (SeeTable 5).

TABLE 5 Cross-reactivity Results Comparison of Three Novel MonoclonalAntibodies for UTIs versus a Standard Polyclonal Antibody using purifiedprotein standards. Crossreactivity Result for Proteins Tested AntibodyHSA THP α-1MG α-1ACT Mab 421-3G5 none none none none Mab 421-5G8 nonenone none none Mab 420-5D11 none = approx. none none 100%Rb-anti-Uristatin none >=50% none none

The broad objective of the invention is to provide a method forconveniently testing the urine of medical patients to identify thepresence of diseases by their association with inhibitory urinarytrypsin inhibitors (UTI). It has been previously noted that the presencein urine of UTIs indicates disease. Heretofore, it has not been possibleto relate particular UTIs with certain diseases since the UTIs generallywere measured in the aggregate, rather than individually. The presentinventors have developed monoclonal antibodies using samples of UTIsisolated from the urine of known renal disease patients and associatedthe monoclonal antibodies with the various fragments of thenon-inhibitor precursors of UTIs. As the examples have shown, themonoclonal antibodies have been found to have varying responses to thesamples of UTIs, which enable one to identify their preference forcertain of the UTIs. Additional investigation by mass spectroscopy hasextended the information available about the composition of the UTIsamples and the response of the monoclonal antibodies to them. Oneimportant finding has been the discovery that a lower molecular weightUTI fragments, at about 2.8, 3.5, 3.9 and 5.4 kDa was an importantcomponent in the total UTIs observed in samples. That result was notseen in SDS-Page testing as only the proteins with the highestconcentration are visualized and consequently the results of ELISAtesting with the monoclonal antibodies could not be completelyinterpreted. Since the use of mass spectroscopy in routine clinical workis impractical, the present inventors have applied the informationobtained from mass spectroscopy to interpretation of the results ofELISA tests, which may be more readily used in clinical settings.

The proposed procedures for testing urine samples may be outlined asfollows:

-   1. Prepare monoclonal antibodies against known sources of one or    more UTIs.-   2. Test the monoclonal antibodies against the known sources of UTIs    and select those that are found to have a preference for UTIs having    known molecular weights.-   3. Add the selected monoclonal antibodies to a sample of urine    containing unknown amounts of UTIs and determine which UTIs are    present and their relative amounts.-   4. Correlate the UTIs determined in step 3 with a disease associated    with the measured distribution of UTIs.

The UTIs that have been identified by SDS-PAGE methods and correlatedwith ELISA tests include fragments of the I-α-I and P-α-I non-inhibitorsthat have typical average molecular weights of about 66 kDa (AMBK), 30kDa (Bikunin or HI-30), 17 kDa (Uristatin), 5 kDa (Uristatin-1), 8 kDa(Uristatin-2) and ranges defined around these averages due tofragmentation, variation and aggregation. In particular, it was foundalso that smaller fragments having molecular weights of about 2 to 5 kDawere present. Those were designated as fragments of Uristatin-1 and 2.It now appears from mass spectroscopy that fragments at about 2.8, 3.5,3.8 and 5.0 kDa are present in samples and bound by antibodies, but notclearly shown by the SDS-PAGE results.

The monoclonal antibodies appeared to be grouped in their response tosamples containing known distributions of particular UTIs. That is, someappeared to bind to UTI samples containing significant amounts of 2-12kDa fragments (Uristatin 1 or 2), while others appeared to bind to UTIsamples containing a large fraction of AMBK, Bikunin or the Uristatinfractions of 62, 30 and 17 kDa. Still others bound to Bikunin ,Uristatinfractions and THP common sequence. Three monoclonal antibodies wereselected as characteristic of their groups and those antibodies weredeposited in the ATCC depository. Other monoclonal antibodies havingsimilar properties are identified and their performance reported in theexamples for further information.

The advantage of identifying monoclonal antibodies having a preferencefor certain UTIs is of course that it opens the possibility ofdistinguishing certain diseases or other medical problems with aparticular distribution of UTI fragments. Certain antibodies may bespecific for binding certain UTI fragments or for certain combinationsof fragments. Then, by using those monoclonal antibodies in ELISA orother such tests it will be possible to identify which of the UTIs arepresent and their relative proportions.

1. A method of assaying a biological fluid for urinary tryspininhibitors (UTI) selected from the group consisting of uristatin,uristatin-1, uristatin-2 and THP comprising contacting a sample ofbiological fluid with a monoclonal antibody against purified uristatinand detecting said UTIs bound by said monoclonal antibody.
 2. A methodof claim 1, wherein said monoclonal antibody is secreted by a hybridomaselected from the group consisting of ATCC 421-5G8.1A8.5C1, ATCC420-5D11.5G8.1E4, and ATCC 421-3G5.4C5.3B6.
 3. A method of claim 2,wherein said monoclonal antibody is secreted from hybridoma ATCC421-5G8.1A8.5C1.
 4. A method of claim 2, wherein said monoclonalantibody is secreted from hybridoma ATCC 420-5D11.1E4.
 5. A method ofclaim 2 wherein said monoclonal antibody is secreted from hybridoma ATCC421-3G5.4C5.3B6.
 6. A method of claim 2 wherein said UTIs are detectedin an immunoassay.
 7. A method of claim 6 wherein said immuno assay isselected from the group consisting of MIC (microparticle captureimmunoassays, LAI (latex agglutination inhibition), IC (solid phasechromatographic), RIA (radio immunoassays), ELISA (enzyme linkedimmunoabsorbent assay), BIA (enzyme linked assays), FIA (fluorescencelinked assays), LIA (luminescence linked assays), CLA (chemiluminescenceassays), OA (optical color label assays), EST (electrochemical signaltransducers) and rare earth metals label assays.
 8. A method of claim 1wherein said biological fluid is urine.
 9. A method of claim 1 whereinsaid biological fluid is blood plasma.